Synthetic GTF chromium material for decreasing blood lipid levels and process therefor

ABSTRACT

A novel product obtained by reacting an alkali metal salt of nicotinic acid with a trivalent chromium salt and having glucose tolerance factor activity, a process for its production and a method for its use, are disclosed.

RELATED APPLICATION DATA

This is a divisional of application Ser. No. 186,149, filed Apr. 26,1988 that application is a continuation-in-part of commonly-owned andco-pending application Ser. No. 512,111, filed July 8, 1983 abandoned.

TECHNICAL FIELD

The present invention relates generally to glucose metabolism, and moreparticularly to the production and use of a novel chromium-containingproduct which has glucose tolerance factor (GTF) activity.

BACKGROUND OF THE INVENTION

Although the physiological mechanism is not completely understood, ithas been reported that animals placed on a purified, chromium-free dietfor several weeks displayed greatly impaired glucose tolerance, i.e. theability to maintain blood glucose at normal levels. It was found that adiet containing Brewer's yeast would eliminate this impairment and bloodglucose levels would return to normal.

The presence of chromium as an organic salt in foods was also found toincrease glucose oxidation in humans, particularly when extracts ofBrewer's yeast containing chromium were added. In addition, oraladministration of such material to a diabetic individual was found toinfluence the pancreas to produce normal amounts of insulin.

The relationship of chromium content in food and its effects on glucoseoxidation activity are discussed, for example, in Toepfer, et al.,"Chromium Foods in Relation to Biological Activity," J. Agr. Food. Chem.21:69 (1973).

These and other findings led to an interest in Brewer's yeast as asource of a naturally occurring glucose tolerance factor (GTF) by earlyworkers in the field of trace mineral research. A discussion of thehistory of GTF research can be found in McCarthy, et al., "High-ChromiumYeast and Glucose Tolerance Factor," J. Prevention Medicine 2 (1983).Considerable research has been directed towards concentrating thechromium content of Brewer's yeast to a commercially feasible amount.See, for example, U.S. Pat. No. 4,343,905.

Glucose tolerance factor is believed to be a complex of trivalentchromium with two moles of nicotinic acid (niacin) and at least one moleof an amino acid. For example, as suggested by Mertz, et al. in "PresentKnowledge of the Role of Chromium," Fed. Proc. 33(11):2275-2280 (1974),it is possible that the structure for natural GTF could be derived fromthe structure for a tetraaquo-dinicotinato Cr-complex, i.e., ##STR1##wherein the molecules of water are replaced by amino acid ligands.

Thus, although the exact structure of GTF is not known at this time, onepossible structure might be as follows: ##STR2##

Alternatively, a recent patent application, published as WO 87/03200,reports the isolation and purification from yeast of a compound with GTFactivity which is a quinoline derivative having a molecular weight of174 and a structural formula as follows: ##STR3## wherein at least one Rgroup is hydrogen and the two non-hydrogen, non-identical R groups are--OCH₃ or --NH₂. This report includes reference to additional researchefforts which cast doubt on the role of chromium and chromium-nicotinicacid-amino acid complexes in yeast GTF activity.

Attempts have also been made to synthesize trace metal complexes whichexhibit GTF activity. For example, U.S. Pat. No. 4,242,257 discloses amaterial exhibiting GTF activity which is obtained by complexing cobaltwith nicotinamide in a ratio of 1:2, followed by acidification of thecomplex and reduction with glutathione. Similarly, the synthesis ofchromium-nicotinic acid complexes, which exhibit GTF activity, byrefluxing chromium chloride in alcohol for periods of up to about 16 to24 hours with nicotinic acid and several amino acids has been disclosed.See, for example, Toepfer, et al., "Preparation of Chromium-ContainingMaterial of Glucose Factor Activity from Brewer's Yeast Extracts and bySynthesis", J. Agr. Food Chem. 25(1):162-166 (1977).

There has also been a report of a green chromium complex withpyridine-3-carboxylic acid (nicotinic acid) which was soluble in commonorganic solvents, including water, acetone, etc. Chatterjee, B., "DonorProperties of Pyridine and Quinoline Carboxylic Acids: Pyridine andQuinoline Carboxylato Complexes of Chromium(III)," J. Indian Chem. Soc.53:1212-1213 (1976). The report does not disclose any use for thecomplex.

While the prior art techniques of extracting and/or concentratingchromium GTF from Brewer's yeast have been reasonably successful, therequired processing normally is rather complex. This makes the productexpensive when used for chromium supplementation of chromium deficientdiets or for individuals otherwise requiring chromium supplementation.This problem has not been overcome by synthetically prepared GTFmaterials because, for the most part, synthetic GTF materials are oflimited activity, are formed in relatively poor yields, and/or arehighly unstable.

Accordingly, it is an object of the present invention to produce asynthetic GTF material which is stable over extended periods of time.

It is another object of the invention to produce a synthetic GTFchromium complex which can be easily prepared in high yields underrelatively mild conditions.

Yet another object of the invention is to produce a non-polar chromiumGTF material which would be transported actively at the sites ofabsorption of nicotinic acid.

Still another object is to provide a chromium GTF material which can bemodified easily by an individual's metabolic system to the appropriateisomeric form, or which exhibits biological activity on its own withoutneed for any such modification.

Another object is to prepare a chromium GTF material which is yeast-freeand which does not require the use of yeast in its synthesis.

DISCLOSURE OF THE INVENTION

These and other objects and advantages are accomplished in accordancewith the present invention by the disclosure of a syntheticchromium-nicotinate GTF material and a process for its production.

In accordance with one aspect of the invention, a chromium-nicotinateGTF material is synthesized under relatively mild conditions using analkali metal salt of nicotinic acid and a trivalent chromium salt as thestarting materials to produce a chromium-nicotinate GTF material in highyields.

In accordance with another aspect of the invention, achromium-nicotinate GTF material is provided which displays activitycomparable to natural glucose tolerance factor, together with a processfor its production and a method for its use.

BRIEF DESCRIPTION OF THE DRAWING

The figure is a graphic representation of the UV-V.3 spectra of 10 mg/mLchromium chloride and a complex of the present invention.

MODES OF PRACTICING THE INVENTION

In accordance with one aspect of the invention, a process forsynthesizing a chromium-nicotinate GTF material is provided by using analkali metal salt of nicotinic acid and a trivalent chromium salt as thestarting materials.

In preparing the chromium-nicotinate GTF material of this invention, adissociable form of alkali metal, such as an alkali metal hydroxide, andnicotinic acid are first reacted to form the alkali metal salt ofnicotinic acid. Thereafter the nicotinic acid salt is reacted with thetrivalent chromium salt to form the desired GTF product. Alternatively,the alkali metal salt of nicotinic acid is conveniently obtained fromother sources and reacted with the chromium salt to form the desired GTFproduct.

The reactions normally would be carried out in a polar solvent system,such as an aqueous or alcohol solvent, and would require only mildreaction temperatures, e.g., on the order of from about 5° C. to about60° C., preferably from about 10° C. to about 40° C. While the statedtemperature range is not a rigid requirement, and while temperaturessomewhat above or below the stated range could be employed, the addedcost of cooling and/or heating the reactants without commensuratebenefits in speed or yield is considered to render the use oftemperatures outside the stated range less desirable.

If used to prepare the nicotinic acid salt, the dissociable alkali metalmay comprise any of a number of compounds containing an alkali metal,including an alkali metal hydroxide, such as sodium hydroxide, potassiumhydroxide, lithium hydroxide, or magnesium hydroxide. Alternatively, analkali metal carbonate or bicarbonate, e.g. containing sodium,potassium, lithium or magnesium, may be used in place of the alkalimetal hydroxide.

However, for reasons of economics and ease of handling, it is presentlypreferred to use sodium hydroxide as the dissociable alkali metal.Sodium hydroxide may be used in the form of food or reagent gradepellets (J. T. Baker Co.) or as a solution. Again, however, tofacilitate the preparation of a complex for a food additive, it ispreferable to use food grade pellets of sodium hydroxide.

In forming a representative salt of nicotinic acid, the dissociablealkali metal can be added to a solution containing the nicotinic acidand allowed to react without further effort. Such a solution willgenerally, but not necessarily, be prepared in the same solvent selectedfor the process of the invention.

The trivalent chromium salts presently preferred for use in the presentinvention include any readily soluble and pharmaceutically acceptablesalts, such as, for example, chromic chloride, chromic sulfate, chromicacetate, and the like, which do not interfere with the formation of thedesired chromium GTF material and which do not form toxic by-products.In a presently preferred embodiment, CrCl₃ . 6H₂ O (chromium chloridehexahydrate) or Cr(CH₃ COO)₃ . 6H₂ O (chromium acetate hexahydrate)would be used for the chromium salt.

Solvents which have been found useful in the process of the presentinvention include polar solvents, such as water and alcohols, typicallysaturated or unsaturated alkyl chains of from one to 10 carbon atoms,more usually one to six carbon atoms. Such alcohols will have thegeneral formula

    R.sub.1 --OH

wherein R₁ is an organic alkyl group of the form CH₃ (CH₂)_(n) -- ineither straight chain or branched form and n is a whole number of from 0to 9. Especially useful solvents have been found to include water andmethyl, ethyl, propyl, isopropyl, butyl and isobutyl alcohols, andmixtures thereof.

It will be readily understood, however, that while certain parameterswithin the following protocols may need to be altered slightly inaccordance with known principles, numerous additional polar solvents maybe employed in the practice of the present invention. For example, manypolar organic solvents, e.g. chlorinated hydrocarbons, ethers, ketones,hydrocarbons and alcohols, either alone or in the presence of water oralcohols, can also find use as solvents in performing the process of thepresent invention.

The reaction between the chromium salt and the alkali metal salt ofnicotinic acid, e.g., sodium nicotinate, takes place quite rapidly andwill be evidenced by a distinct color change and subsequentprecipitation of the chromium GTF material. For example, when an aqueousreaction medium is employed, the green color imparted to the reactionmedium by the chromium chloride salt disappears in a matter of seconds,usually in less than about 60 to 120 seconds, and an intenselypurple-colored material precipitates from solution. This precipitatedmaterial is the chromium-nicotinate GTF product of the invention, whichis believed to contain significant amounts of chromium trinicotinateand, most probably, some chromium dinicotinate mono-chloride, -acetate,or -sulfate, etc., depending upon the anion contained in the chromiumsalt reactant.

The present invention contemplates a stable syntheticchromium-nicotinate GTF material which has been prepared by reactionbetween an alkali metal salt of nicotinic acid, such as sodiumnicotinate, and a trivalent chromium salt, such as chromium chloride.The crude chromium-nicotinate GTF material comprises at least asubstantial portion of a trinicotinic chromium complex (chromiumtrinicotinate) and the reaction sequence may be represented by thefollowing equations: ##STR4##

The exact structure of the chromium trinicotinate material is not known,nor is its exact structure critical to the present invention. However,it is believed that the structure of the trinicotinate complex may beillustrated generally as follows: ##STR5##

It is likely that the reaction illustrated in equation (IV) does not goto completion and that some dinicotinate complex is formed, thusresulting in a product containing both chromium trinicotinate andchromium dinicotinate mono-anion (e.g., chloride, sulfate or acetate,etc.). It is also possible that some chromium mononicotinate di-anionmight be present in the crude reaction product, or that a poly-nuclearspecies such as dichromium pentanicotinate may be formed. However, it isbelieved, although not required, that chromium trinicotinate is thepredominant reaction product and exhibits the GTF activity in thepresent chromium-nicotinate GTF material.

Subsequent to its formation, the product of this invention can be dried,for example at temperatures from about 10° C. to about 150° C.,preferably from about 20° C. to about 100° C., and at less than 100%relative humidity. In addition, optionally the product can be washedsubsequent to its formation in order to remove soluble unreactedcompounds which may have become entrapped in the product during theprecipitation from solution. Desirable wash solutions would includewater and other solvents in which the chromium-nicotinate GTF materialis substantially insoluble.

Products of the present invention are shown to have glucose tolerancefactor activity in the intact mammal. Furthermore, products of thepresent invention may demonstrate other effects in vivo, such asincreased glucose oxidation levels. Such products shown to have theabove recited physiological effects can find use in numeroustherapeutical applications such as, e.g., increasing glucose tolerancein chromium deficient patients and potentiating the effect of insulin indiabetics. Thus the products of the invention, and compositionscontaining them, can find wide use as therapeutic agents.

Thus the present invention also provides compositions containing aneffective amount of compounds of the present invention which may, alone,serve to provide the above-recited therapeutic benefits. Suchcompositions can also be provided together with physiologicallytolerable liquid, gel or solid diluents, adjuvants and excipients, asare known in the pharmaceutical art, for example, as described inRemington's Pharmaceutical Sciences (16th ed., 1980).

These products and compositions can be administered to mammals forveterinary use, such as with domestic animals, and clinical use inhumans in a manner similar to other therapeutic agents. In general, thedosage required for therapeutic efficacy will range from about 0.01 to1000 μg/kg, more usually 0.1 to 1000 μg/kg of the host body weight,depending upon such factors as the blood sugar reducing activity of thechromium-nicotinate GTF material, and any particular requirements of thepatient. Alternatively, dosages within these ranges can be administeredby constant infusion over an extended period of time, usually exceeding24 hours, until the desired therapeutic benefits have been obtained.

Typically, such compositions are prepared for oral administration,either as liquid solutions or suspensions to be administeredindependently or as a food supplement. Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin,cellulose, magnesium carbonate, and the like. These compositions contain10-95% of active ingredient, preferably 25-70%, and take the form ofsolutions, suspensions, tablets, capsules, sustained releaseformulations, or powders.

Additional formulations which are suitable for other modes ofadministration can include suppositories, intranasal aerosols, and, insome cases the compositions are administered parenterally, by injection,for example, either subcutaneously or intravenously. As injectables, theactive ingredient is often mixed with diluents or excipients which arephysiological tolerable and compatible with the active ingredient.Suitable diluents and excipients are, for example, water, saline,dextrose, glycerol, or the like, and combinations thereof. In addition,if desired the compositions may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, stabilizing or pHbuffering agents, and the like. For suppositories, traditional bindersand excipients may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5-10%, preferably 1-2%.

In addition to the products of the present invention which display GTFactivity, products of the present invention can also be employed asintermediates, for example in the further purification of such usefulcompounds.

Experimental

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, all weights are given ingrams (g), milligrams (mg) or micrograms (μg), all volumes are given inliters (L) or milliliters (mL) and all wavelengths are given inmillimicrons (mμ) unless otherwise indicated.

EXAMPLE 1

In order to illustrate the synthesis of the chromium-nicotinate GTFmaterial of the present invention, a presumptive trinicotinate complexwas prepared as follows:

A solution of 3,673 g of nicotinic acid (niacin) USP in 4,540 gdistilled water was mixed by stirring. Into this mixture was added 1,224g of food grade sodium hydroxide pellets (J. T. Baker Co.) withstirring, thereby forming a solution containing an alkali metal salt ofnicotinic acid.

In a separate container, 2,724 g of reagent grade chromium chloridehexahydrate (CrCl₃ . 6H₂ O) was added to 1,589 g of distilled water andstirred until completely dissolved, to provide a solution containing atrivalent chromium salt.

The chromium chloride solution, which had a green color, was then addedslowly to the nicotinic acid salt solution with rapid stirring. Themixture formed an intense purple complex which precipitated from thegreen chromium solution. The precipitate was collected by filtration andplaced on trays to dry.

The intense color change from green to purple and the subsequentprecipitation denoted the chemical change of the chromium ion to thecomplex. This color shift occurred because the atomic orbitals ofchromium became occupied, and the wavelength of light absorbed shiftedaccordingly. The complex precipitated because it had no charge,indicating that the negatively charged nicotinate ions were complexedwith the Cr⁺ ion.

The presence of the complex in the chromium-nicotinate GTF material wasfurther demonstrated by the following analyses:

Chemical Analysis

1. Moisture=6%-9%

2. Niacin Content=50%-66%

3. Chromium Content=8%-10%

4. Sodium Content=8%-11%

5. Chloride Content=12%-16%

Elemental Analysis

The content of certain elements in a representative sample of theproduct showed:

Carbon 43.21%

2. Hydrogen 2.77%

3. Chromium 12.9%

4. Nitrogen 8.33%

5. Chloride 3.31%

Polarographic Analysis

A definite shift in the E-1/2 was observed. However, good qualitativeresults were not possible due to the fact that niacin is reduced atabout the same voltage range.

UV -V.3 Spectra

As shown in the Figure, two absorbance maxima were observed for chromiumchloride in the visible range at 637 mμ and 461 mμ. When the complex wasformed, the absorbance maxima shifted to 574 mμ and 414 mμ.

Infrared Spectroscopy

Samples of the chromium-nicotinate GTF material prepared according toExample 1 were washed several times with analytical grade water anddried at 120° C. for three hours. Chromate and nicotinic acid sampleswere also analyzed by IR spectroscopy. The samples were suspended in athree percent potassium bromide solution and placed between sodiumchloride prisms.

As an absorbance control, a chromium chloride blank was made by diluting158 mg of CrCl₃ . 6H₂ O to 100 mL with ethanol to give a kelly greencolored solution. The visible light absorbance maxima were 628 mμ and455 mμ.

The results of the IR analysis showed a single asymmetric stretch peakin the range of 1630 reciprocal wave numbers.

EXAMPLE 2

In order to demonstrate the use of alternative starting materials toform the product of the present invention, 290 mg of nicotinic acid and249 mg of sodium carbonate were dissolved into 50 mL of water and the pHwas adjusted to 7.0 with 0.5M NaOH. Then 158 mg of CrCl₃ . 6H₂ O wasdissolved in 50 mL of ethanol and the nicotinate mixture was stirredinto the chromium chloride solution. A purple complex formed withcontinued stirring.

The absorbance maxima were measured on a Perkin Elmer 200 ultra-violetvisible spectrophotometer and found to be at 568 mμ and 424 mμ. Thepurple complex precipitate, when dried, formed a crystalline powdersubstantially insoluble in ethanol or water.

EXAMPLE 3

In order to demonstrate the use of alternative polar solvents in theformation of the product of the present invention, thechromium-nicotinate GTF material was synthesized generally in accordancewith the procedure set forth in Example 1, in which the solvent wasdistilled water, and was also prepared employing ethanol, anhydrousalcohol and methanol as solvents.

In each case, 36.73 g nicotinic acid (niacin) USP was added to 45.40 gof the appropriate solvent until a homogeneous mixture was formed. Next,17.46 mL of NaOH (70% solution) (12.24 g NaOH by weight) was added toform the sodium salt of nicotinic acid.

In a separate container, 27.24 g CrCl₃ . 6H₂ O (reagent grade, J. T.Baker Chemical Co.) was mixed with 15.9g of the appropriate solvent andstirred until the CrCl₃ was completely dissolved. The chromium chloridesolution was then slowly added into the sodium nicotinate solution.

The mixture was then examined for the formation of precipitate. Sampleswere taken and dried at 100° C. overnight, washed with distilled water,and dried at 100° C. for two hours.

This GTF material was then tested for color. The samples of complexproduced in accordance with the present invention in ethanol, anhydrousalcohol, methanol and H₂ O each formed purple-blue to purple-greyprecipitates, which yielded the same color powder upon drying. Thesolubility profiles of these complexes are present in Table I.

EXAMPLE 4

In order to demonstrate the importance of the nicotinic acid salt in theformation of a product in accordance with the present invention, 36.73 gof nicotinic acid (niacin) USP was dissolved in 47 mL of anhydrousalcohol. In a separate container, 27.24 g CrCl₃ . 6H₂ O was mixed with16 g of anhydrous alcohol. The chromium chloride solution was slowlymixed with the niacin solution and stirred for two hours at roomtemperature. The solution did not produce a precipitate.

The product of this example formed a greenish solution which did notprecipitate under the test conditions. The dried and washed sampleformed a greenish powder.

The samples of complex produced in Examples 1 through 4 were tested forsolubility in H₂ O, methanol, ethanol and anhydrous alcohol. The resultsof the solubility testing are presented in Table I.

                  TABLE I                                                         ______________________________________                                                EXAMPLE 4  GTF-                                                               PRODUCT    ALCO-    GTF-   GTF-                                       SOLVENT (ALCOHOL)  HOL      H.sub.2 O                                                                            METHANOL                                   ______________________________________                                        H.sub.2 O                                                                             Soluble    Not Sol. Not Sol.                                                                             Not Sol.                                   Methanol                                                                              Not Sol.   Not Sol. Not Sol.                                                                             Part. Sol.                                 Ethanol Part Sol.  Not Sol. Not Sol.                                                                             Not Sol.                                   Anhydrous                                                                             Part Sol.  Not Sol. Not Sol.                                                                             Not Sol.                                   Alcohol                                                                       ______________________________________                                    

It has been stated that a true GTF material should have the followingcharacteristics:

1. It must improve glucose tolerance;

2. It must be transported across the placenta;

3. The metabolism of adipose tissue must be increased (higher rate offat and glucose burning); and

4. It must be metabolized differently than chromium chloride and becomepart of a different biological pool for this element.

Accordingly, samples of the chromium nicotinate reaction product formedin Example 1 were tested to demonstrate their use as a GTF material asoutlined above.

EXAMPLE 5

A synthetic rat food was prepared as a 2.2 g tablet with the followingformulation:

    ______________________________________                                        Vitamin A                                                                             30       IU     Cu          0.0005 mg                                 D3      0.4      IU     Zn          0.0007 mg                                 Vitamin E                                                                             1.0      IU     Mn          0.0006 mg                                 Vitamin C                                                                             0.1      mg     Mo          0.000025                                                                             mg                                 Folic   0.004    mg     K           5      mg                                 B1      0.0l2    mg     Na          6      mg                                 B2      0.006    mg     Se          0.00005                                                                              mg                                 B3      0.02     mg     Inositol    1      mg                                 B6      0.005    mg     Paba        0.005  mg                                 B12     0.000006 mg     V           0.000025                                                                             mg                                 Biotin  0.0003   mg     EMDEX ®.sup.1                                                                         900    mg                                 Pantothenic                                                                           0.01     mg     Casein      900    mg                                 Acid                    Sunflower Oil                                                                             200    mg                                 Choline 0.03     mg     SYLOID ®.sup.2                                                                        20     mg                                 Ca      1.0      mg     Stearic Acid                                                                              20     mg                                 Iodine  0.0001   mg     SOLKA-FLOC ®.sup.3                                                                    200    mg                                 Fe      0.008    mg                                                           Mg      0.4      mg                                                           ______________________________________                                    

Fifteen male albino rats, each weighing between about 180 to 200 g, wereplaced on a diet of the above synthetic rat food for two weeks. The ratswere then divided into three groups of five subjects each. Group I wasfed only the above synthetic rat food. Group II was fed the syntheticrat food together with 10 ppm of the chromium-nicotinate GTF material ofExample 1 added to the drinking water. Group III was fed the syntheticrat food with 10 ppm of chromium as chromium chloride added to thedrinking water. This diet regime was maintained for six weeks.

Each subject was weighed every two weeks. The growth rates of thesubjects from each group demonstrated that the GTF chromium product hada marked effect on the total body weight and total fat content of therats. At the end of six weeks on the diet, the subjects were sacrificed,dissected and the organs were assayed for chromium content. The resultsof the assay for chromium were as presented in Table II.

During dissection, it was noted that the subjects in Groups I and IIIhad large amounts of adipose tissue beneath the skin and laced in theintestines. The effects of the GTF chromium product in preventing thebuildup of fatty tissue in Group II subjects were almost immediate.

The growth rates of subjects from Groups I and III were almost parallel,until the toxicity of chromium chloride caused a decrease in the weightof the animals in Group III. The observed toxicity apparently stems fromrapid chromium chloride build up in the kidneys.

                  TABLE II                                                        ______________________________________                                        Body Organ Analysis - Chromium Content                                        GROUP I        GROUP II     GROUP III                                         μg/gm  μg/organ                                                                            μg/gm                                                                              μg/organ                                                                          μg/gm                                                                            μg/organ                           ______________________________________                                        Heart 0.243   0.310    0.506 0.582  0.601 0.685                               Liver 0.267   3.95     0.282 3.14   0.406 4.199                               Spleen                                                                              0.382   0.253    0.950 0.698  0.650 0.439                               Kidney                                                                              0.184   0.341    0.443 0.652  1.136 1.823                               Muscle                                                                              0.206   --       0.179 --     0.239 --                                  ______________________________________                                    

In addition to the organ analysis, blood was collected from eachsubject, and the serum was separated from the red blood cells. The serumwas assayed for glucose, triglycerides and cholesterol. The results ofthe serum analysis were as presented in Table III.

                  TABLE III                                                       ______________________________________                                        Serum Analysis                                                                        Glucose  Cholesterol                                                                             Triglycerides                                              mg/DL    mg/DL     mg/DL                                              ______________________________________                                        Group I   381        53        86                                             Control                                                                       Group II  147        48        49                                             GTF                                                                           Group III 175        52        64                                             CrCl.sub.3                                                                    ______________________________________                                    

This example showed that the GTF chromium is metabolized differentlythan chromium chloride and that it flows to a different biological pool.The GTF chromium also increases the rate of fat metabolism of adiposetissue.

EXAMPLE 6

In order to demonstrate that the chromium-nicotinate GTF materialprepared in accordance with Example 1 will be transported across theplacenta, 15 timed pregnant albino rats were divided into three groupsof five subjects each. These rats were allowed to equilibrate for oneweek, and were then weighed and placed on the diet regime of Example 5.Group I was the control group and was fed the synthetic rat food ofExample 5. Group II was fed the synthetic rat food with thesupplementation of 10 ppm of the chromium-nicotinate GTF material ofExample 1 added to the drinking water. Group III was fed the syntheticrat food supplemented with 10 ppm chromium as chromium chloride in thedrinking water.

At the end of one week, the subjects were weighed, sacrificed, dissectedand the organs and fetuses were analyzed as described in Example 5. Theresults of the analyses were as presented in Tables IV, V and VI

                  TABLE IV                                                        ______________________________________                                        Organ Analysis - Chromium Content                                             GROUP I        GROUP II     GROUP III                                         μg/gm  μg/organ                                                                            μg/gm                                                                              μg/organ                                                                          μg/gm                                                                            μg/organ                           ______________________________________                                        Heart 0.38    0.372    0.37  0.368  0.34  0.347                               Liver 0.42    5.67     0.29  3.48   0.30  4.034                               Muscle                                                                              0.24    --       0.28  --     0.37  --                                  Kidney                                                                              0.24    0.312    0.33  0.441  0.50  0.758                               Spleen                                                                              0.45    1.478    0.47  0.289  0.63  0.378                               Pla-  0.24    0.218    0.34  0.322  0.49  0.389                               centa                                                                         Fetus 0.17    810/     0.17  1,049/ 0.17  862/                                I             fetus          fetus        fetus                               Fetus 0.17    810/     0.18  1,049/  0.167                                                                              862/                                II            fetus          fetus        fetus                               ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        Serum Analysis                                                                            Group I Group II  Group III                                       ______________________________________                                        Glucose mg/DL 307       222       236                                         Cholesterol mg/DL                                                                           80        61         93                                         Triglycerides mg/DL                                                                         69        76        104                                         ______________________________________                                    

                  TABLE VI                                                        ______________________________________                                        Weight Analysis                                                                         Group I   Group II Group III                                        ______________________________________                                        Mean Initial Weight                                                                       309.8       310      309.2                                        Mean Final Weight                                                                         303         278      296                                          Percent Change                                                                            -2.1%       -10.3    -4.3                                         ______________________________________                                    

This example showed that over the course of one week thechromium-nicotinate GTF material of the present invention wastransported across the placenta, achieving a 30% increase in fetaltissue chromium content over the control group and a 21% increase overthe chromium chloride group.

It is interesting that the chromium-nicotinate GTF material may notachieve high tissue levels, as it is not concentrated in the tissues andhas a very low toxicity. However, it does exhibit biological activity.This indicates that the GTF product acts differently in the body, andenters a different biological pool, from chromium.

The effect of GTF on fat metabolism is dramatic, and total body fat isprobably the most sensitive indicator of the activity of GTF chromium.Again, the chromium chloride showed a high retention rate in thekidneys.

EXAMPLE 7

The weight and organ analysis protocols of Example 6 were repeated on asecond set of subjects, except that the animals were supplemented forten days rather than seven, with the results displayed in Tables VII andVIII:

                  TABLE VII                                                       ______________________________________                                        Organ Analysis - Chromium Content                                             GROUP I        GROUP II     GROUP III                                         μg/gm  μg/organ                                                                            μg/gm                                                                              μg/organ                                                                          μg/gm                                                                            μg/organ                           ______________________________________                                        Heart 0.359   0.291    0.256 0.250  0.322 0.317                               Liver 0.238   2.908    0.375 4.684  0.277 3.096                               Muscle                                                                              0.262   --       0.371 --     0.249 --                                  Kidney                                                                              0.337   0.440    0.324 0.439  0.816 1.052                               Spleen                                                                              0.355   0.233    0.352 0.242  0.470 0.308                               Pla-  0.324   0.252    0.364 0.232  0.338 0.231                               centa                                                                         Fetus 0.349   1.269    0.559 1.665  0.229 1.142                               ______________________________________                                    

                  TABLE VIII                                                      ______________________________________                                        Weight Analysis                                                                         Group I   Group II Group III                                        ______________________________________                                        Mean Initial Weight                                                                       331         343      307                                          Mean Final Weight                                                                         301         311      262                                          Percent Change                                                                            -9.1%       -9.3%    -14.7%                                       ______________________________________                                    

This example verified the improved transport of the chromium-nicotinateGTF material of the present invention over chromium chloride andreconfirmed the toxicity of chromium chloride in the kidney.

In the first pregnancy test (Example 6) the reduced level of body fatwas demonstrated where body weights were determined after delivery andbefore dissection; an accurate determination method. In this example,the body weights were determined before dissection, which did not allowfor the weight of the fetuses or fluid retention. However, the weightchange was found to be approximately the same for Group I and Group II,and both Groups I and II were less than Group III.

It is apparent from the foregoing examples that the chromium-nicotinateGTF material of the present invention exhibits the characteristics of atrue GTF complex. Moreover, since the examples illustrate a reductionand/or control of adipose tissue and serum lipid levels in the rats sotreated, and since this is a well-known characteristic of prior art GTFcompounds, it would be expected that similar effects would be noted onhumans treated with the present chromium-nicotinate GTF material. See,for example, Example 2 of U.S. Pat. No. 4,343,905, which is incorporatedherein by reference.

When the present invention is used for chromium supplementation inhumans the applicable daily dosage for oral administration would dependon the degree of chromium supplementation needed, but may vary from aslittle as 4 μg to as much as about 500 to 1,000 μg, based on thechromium content of the chromium-nicotinate GTF material. Suchsupplementation could be made by adding the material to the individual'sfood or drink, or by administering one or more tablets containing theappropriate levels of the chromium-nicotinate GTF material.

Tablets containing the GTF material of the invention may be prepared,e.g., by dry-mixing 500 mg of EMDEX®, 5 mg magnesium stearate, 2 mgSYLOID® 74 and 2 mg chromium-nicotinate GTF material in accordance withthe present invention. The dry mixture is then pressed into tablets ofabout 7/16 inch diameter. A 2 mg portion of the present GTF materialprovides about 200 μg of chromium, based on present analysis.

The GTF material used is preferably that which has been freed from thesalt and water, such as by washing the chromium-nicotinate GTF materialprepared in accordance with Example 1 with water and filtering with aBuchner® funnel, using a Whatman® No. 4 filter paper. The filtrate isthen rinsed with two more liquid volumes of deionized water and thefilter cake dried at 100° C. overnight.

The chromium-nicotinate GTF material may be added to foods and drinks bysimply including it into the formulation of the product. For example, itmay be added to bread in an amount of about 20 μg chromium per slice atthe dough-making stage. This level of supplementation would requireapproximately 0.1% of the dough to be the chromium-nicotinate GTFmaterial.

When used in treating humans for the purpose of improving glucosetolerance, the dosage would ordinarily be about 500 to 1,000 μg, basedon chromium, per day. When used for the treatment of hyperlipidemia, thepreferred dosage is from 2 to 4 mg chromium per day, based on thechromium level.

Although the foregoing examples illustrate the preparation and use ofparticular chromium GTF product, it will be appreciated that theteachings of this application encompass broader and other aspects thanrecited in the examples. Accordingly, the present invention should belimited only by the scope of the appended claims.

I claim:
 1. A method of decreasing blood lipid levels in a mammal,comprising administering an effective amount of a chromium-nicotinateGTF material to the mammal, wherein the chromium-nicotinate GTF materialis obtained by reacting an alkali metal salt of nicotinic acid with apharmaceutically acceptable trivalent chromium salt.
 2. The method ofclaim 1, wherein the chromium-nicotinate GTF material is prepared byreacting sodium nicotinate with trivalent chromium chloride.
 3. Themethod of claim 1, wherein the chromium-nicotinate GTF materialcomprises a mixture of chromium trinicotinate and chromium dinicotinatemonochloride.
 4. The method of claim 1, wherein the chromium-nicotinateGTF material consists essentially of chromium trinicotinate.